Methods and materials for cultivation and/or propagation of a photosynthetic organism

ABSTRACT

The present disclosure provides methods and materials for the cultivation and/or propagation of a photosynthetic organism. Such methods may comprise the use of a lamp assembly that comprises a plurality of circuit boards, each comprising at least three edges, arranged in a substantially spherical shape defining an interior lamp assembly volume, wherein the plurality of circuit boards comprise a first planar surface in contact with the interior lamp assembly volume and an opposing second planar surface comprising light emitting diodes (LEDs); and a barrier that surrounds the plurality of circuit boards forming the substantially spherical shape.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 61/612,001 filed Mar. 16, 2012.

BACKGROUND

Photobioreactors have been described for the use of cultivating alga andgenerally employ shallow lagoons agitated with one or more paddlewheels. Such bioreactors are plagued with problems including poorproduction of algae due to seasonal and daily climatic changes andcontamination. Given that such bioreactors are generally constructed toreceive the sun's daylight light, productivity is limited by intensityof the sun which depends on the photoperiod and the season, among otherfactors.

SUMMARY

Methods and materials are provided for the cultivation including, forexample, propagation of a photosynthetic organism.

The methods disclosed herein may comprise the use of a photobioreactorthat comprises the use of an electromagnetic source in the visible lightspectrum. In one embodiment, the electromagnetic source comprises aplurality of circuit boards. In another embodiment, each circuit boardcomprises at least three edges, arranged in a substantially sphericalshape defining an interior lamp assembly volume. In another embodiment,the plurality of circuit boards arranged in a substantially sphericalshape comprise a first planar surface in contact with the interior lampassembly volume and an opposing second planar surface comprising highintensity lamps including, for example, light emitting diodes (LEDs). Inanother embodiment, the photobioreactor comprises a barrier thatsurrounds the plurality of circuit boards, said barrier having asubstantially spherical, ovoid, egg, cylindrical, rectangular prismic orother similar shape. Such methods may be used to produce compounds(e.g., biomolecules) including, for example, fatty acids,phycobiliproteins such as C-Phycocyanin, allophycocyanin, phycoerythrin,biofuels such as phytol, and other various petrol fuel substitutes.

The present disclosure also provides a photobioreactor for cultivationand/or propagation of a photosynthetic organism. In one embodiment, thephotobioreactor comprises an electromagnetic source in the visible lightspectrum. In another embodiment, the photobioreactor comprises a vesselhaving a wall defining an interior vessel volume. In one embodiment, thevessel is substantially cylindrical, spherical, rectangular prismic orovoid in shape. In another embodiment, the photobioreactor comprises alamp assembly positioned within the interior vessel volume, wherein thelamp assembly optionally comprises a plurality of circuit boards, eachoptionally comprising at least three edges, arranged in a substantiallyspherical or ovoid shape defining an interior lamp assembly volume. Inone embodiment, the plurality of circuit boards each comprise a firstplanar surface in contact with the interior lamp assembly volume and anopposing second planar surface comprising light emitting diodes (LEDs).In one embodiment, the photobioreactor comprises a barrier thatsurrounds the plurality of circuit boards. In one embodiment, thebarrier is substantially cylindrical, spherical, rectangular prismic orovoid in shape.

In some embodiments, which may be combined with any of the above orbelow embodiments, the barrier is cylindrical and comprises acylindrical wall, an upper wall, and a lower wall each defining aninterior tank volume.

In some embodiments, which may be combined with any of the above orbelow embodiments, the vessel is substantially spherical or ovoid.

In some embodiments, which may be combined with any of the above orbelow embodiments, the vessel comprises an opening for a gas inlet.

In some embodiments, which may be combined with any of the above orbelow embodiments, the vessel comprises an opening for a gas outlet.

In some embodiments, which may be combined with any of the above orbelow embodiments, the vessel comprises an opening for wiring the lightsource.

In some embodiments, which may be combined with any of the above orbelow embodiments, the lamp assembly is positioned substantially in thecenter of the vessel.

In some embodiments, which may be combined with any of the above orbelow embodiments, two or more lamp assemblies are positioned in thevessel.

In some embodiments, which may be combined with any of the above orbelow embodiments, the two or more lamp assemblies are positioned atdifferent heights in the vessel.

In some embodiments, which may be combined with any of the above orbelow embodiments, three or more lamp assemblies are positioned in thevessel.

In some embodiments, which may be combined with any of the above orbelow embodiments, the three or more lamp assemblies are positioned atdifferent heights in the vessel.

In some embodiments, which may be combined with any of the above orbelow embodiments, the three or more lamp assemblies are positioned in ahelical arrangement in the vessel.

The present disclosure also provides a light source for use incultivation and/or propagation of a photosynthetic organism. In oneembodiment, the light source comprises: a plurality of circuit boards,each comprising at least three edges. In one embodiment, the circuitboards are arranged in a substantially spherical shape defining aninterior lamp assembly volume, wherein the plurality of circuit boardscomprise a first planar surface in contact with the interior lampassembly volume and an opposing second planar surface comprising lightemitting diodes (LEDs); and a barrier that surrounds the plurality ofcircuit boards forming the substantially spherical shape.

In some embodiments, which may be combined with any of the above orbelow embodiments, the substantially spherical shaped arrangement of theplanar circuit boards has a side devoid of at least one circuit board topermit electrical connectivity. Alternatively the spherical shapedarrangement of the planer circuit boards has an aperture to permitelectrical connectivity.

In some embodiments, which may be combined with any of the above orbelow embodiments, the circuit boards comprise two or more tabs aroundtheir perimeter that form one or more notches that permit the circuitboards to interlock.

In some embodiments, which may be combined with any of the above orbelow embodiments, the circuit boards are pentagon shaped.

In some embodiments, which may be combined with any of the above orbelow embodiments, eleven pentagons are joined to form a dodecahedrondevoid of one side. In another embodiment, twelve pentagons are joinedtogether to form a dodecahedron with an aperture in one or more of thepentagons.

In some embodiments, which may be combined with any of the above orbelow embodiments, the circuit boards are triangular shaped.

In some embodiments, which may be combined with any of the above orbelow embodiments, twenty triangles are joined to form an icosahedrondevoid of one side. In another embnodiment, twenty one triangles arejoined to form an icosahedron with an aperture in one or more triangles.

In some embodiments, which may be combined with any of the above orbelow embodiments, the circuit boards comprise red, white, and blueLEDs.

In some embodiments, which may be combined with any of the above orbelow embodiments, the red, white, and blue LEDs are positioned adjacentto an LED of opposing color. In some embodiments, which may be combinedwith any of the above or below embodiments, the LEDs are pulse widthmodulated.

In some embodiments, which may be combined with any of the above orbelow embodiments, the barrier is plastic.

In some embodiments, which may be combined with any of the above orbelow embodiments, the barrier is substantially spherical or ovoid.

In some embodiments, which may be combined with any of the above orbelow embodiments, the plastic permits transmission of light.

In some embodiments, which may be combined with any of the above orbelow embodiments, the barrier has an opening to permit electricalconnectivity.

In some embodiments, which may be combined with any of the above orbelow embodiments, a void between the barrier and the circuit boardscomprises a fluid for dispersal of heat.

In some embodiments, which may be combined with any of the above orbelow embodiments, the fluid is mineral oil.

The present disclosure also provides methods of producingdocosahexaenoic acid (DHA) comprising: providing one or morephotosynthetic organisms comprising enzymes for generating DHA; addingthe photosynthetic organisms to a vessel of a bioreactor, for example asdescribed herein, comprising a liquid growth media; contacting the oneor more photosynthetic organisms with light emitted from a lampassembly. In one embodiment, the lamp assembly comprises a plurality ofcircuit boards. In another embodiment, each of the circuit boards arearranged in a substantially spherical shape defining an interior lampassembly volume, wherein the plurality of circuit boards comprise afirst planar surface in contact with the interior lamp assembly volumeand an opposing second planar surface comprising light emitting diodes(LEDs). In one embodiment, the lamp assembly comprises a barrier thatsurrounds the plurality of circuit boards forming the substantiallyspherical or ovoid shape. In other embodiments, the plurality of circuitboards are arranged in a shape of any 4-, 6- or 8-sided triangular,planar geometric shape such as a tetrahedron, two-stacked tetrahedrons,an octahedron, or a 20 sided planar geometric shape, such as anicosahedron.

In some embodiments, which may be combined with any of the above orbelow embodiments, the one or more photosynthetic organisms comprisealgae and/or a productive algal culture.

In some embodiments, which may be combined with any of the above orbelow embodiments, two or more algae are provided that have naturalenvironments that are similar in salinity and dissimilar in temperature.

In some embodiments, which may be combined with any of the above orbelow embodiments, the algae are selected from the group consisting ofIsochrysis aff. Galbana, pavlova lutheri, arthrospira platensis,chlorella pyrenoidosa, synechococcus elongates, including naturallyoccurring or genetically modified/recombinant strains of the foregoing.

In some embodiments, which may be combined with any of the above orbelow embodiments, the methods further comprise isolating the DHA fromthe growth media.

The present disclosure also provides methods for storage of a lightenergy comprising: providing one or more photosynthetic organismscomprising enzymes for generating one or more compounds from a lightenergy; adding the one or more photosynthetic organisms to a tank of abioreactor comprising a liquid growth media; contacting the one or morephotosynthetic organisms with light emitted from a lamp assembly, forexample as set forth herein, and producing one or more compounds fromthe light energy. In one embodiment, the lamp assembly comprises: aplurality of circuit boards, each comprising at least three edges,arranged in a substantially spherical shape defining an interior lampassembly volume, wherein the plurality of circuit boards comprise afirst planar surface in contact with the interior lamp assembly volumeand an opposing second planar surface comprising light emitting diodes(LEDs); and a barrier that surrounds the plurality of circuit boardsforming the substantially spherical shape.

In some embodiments, which may be combined with any of the above orbelow embodiments, energy is subsequently released from the one or morecompounds.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary, as well as the following detailed description ofthe disclosure, will be better understood when read in conjunction withthe appended figures. It should be understood that the disclosure is notlimited to the precise arrangements, examples and instrumentalitiesshown.

FIG. 1 shows a schematic of an exemplary spherical-shapedphotobioreactor as described herein.

FIG. 2 shows a schematic of an exemplary dodecahedron shaped lightsource comprised of pentagonal-shaped circuit boards (A), an exemplaryicosahedron shaped light source comprised of triangular-shaped circuitboards (B).

FIG. 3 shows a schematic of an exemplary lamp assembly comprising alight source comprised of pentagonal-shaped circuit boards (A), anexemplary lamp assembly comprising a light source comprised oftriangular-shaped circuit boards (B).

FIG. 4 shows a schematic of the interior of an exemplary verticalbarrel-shaped photobioreactor as described herein.

FIG. 5 shows a schematic of an exemplary vertical barrel-shapedphotobioreactor as described herein.

FIGS. 6A-F show various positions of lamp assemblies within a horizontalbarrel shaped photobioreactor as described herein.

FIG. 7 shows an exemplary rack configuration for horizontal barrelshaped photobioreactors as described herein.

FIG. 8 shows an exemplary rack connection configuration for horizontalbarrel shaped photobioreactors A and B as described herein.

FIG. 9 shows a block diagram of an example control system for managingthe growth of cultures, according to an example embodiment of thepresent invention.

FIG. 10 shows a flow diagram illustrating example procedures to controlthe growth of a culture, according to an example embodiment of thepresent invention.

FIG. 11 shows an illustrative vessel lid (A) and illustrative pebbleconfigurations (B).

FIGS. 12 to 28 are diagrams showing embodiments of lamp assembliesdescribed herein.

DETAILED DESCRIPTION

The present disclosure provides methods and materials for thecultivation and/or propagation of a photosynthetic organism such as analga using a photobioreactor. The photobioreactor provided hereincomprises a lamp assembly that comprises a substantially spherical lightsource positioned in a vessel of the photobioreactor that comprises aliquid medium and a photosynthetic organism. The use of such aphotobioreactor permits unexpectedly high growth and density of thephotosynthetic organism thus maximizing bioconversion efficiencies andproduct yields from the photosynthetic organism. The methods providedherein may be used to produce one or more compounds (e.g., biomolecules)including but not limited to fatty acids such as docosahexaenoic add(DHA), docosapentaenoic acid (DPA), eicosapentaenoic acid (EPA) or otherfatty acids or other compounds such as phycobiliproteins (e.g.C-Phycocyanin, Allophycocyanin, Phycoerythrin, etc), and biofuels suchas phytol and other various petrol fuel substitutes) from thephotosynthetic organism. Additionally or alternatively, such methodsdisclosed herein may be used to provide for storage of a light energy.

Referring now to FIG. 1, a photobioreactor in accordance with anembodiment of the present invention is shown. Photobioreactor 101comprises a vessel 102 (e.g., a tank) for containing a liquid culturemedium 103 for cultivating and/or propagating a photosynthetic organism.

The photobioreactor 101 of the invention is suitable for the culture ofany kind of photosynthetic microorganism, such as a plant cell andunicellular or multicellular microorganisms having a light requirement.As used herein, the term “photosynthetic microorganism” also includesorganisms genetically modified by techniques well known to one skilledin the art.

The liquid culture medium is sometimes referred to herein as an “algal”culture, but it will be appreciated that the photobioreactor may beemployed for the cultivation of any type of photosyntheticmicroorganism.

The vessel 102 may be covered by lid 104. In one embodiment, lid 104 isconstructed of an inert material including, for example, plastics suchas polyvinyl chloride, high-density polyethylene, low-densitypolyethylene and polypropylene. The top rim of the vessel 102 may beformed into a lip which permits lid 104 to be secured (e.g., bolted orclamped) to the top of vessel 102. In addition, the top rim of vesselmay be fitted with a gasket material to provide a liquid and gas-tightseal with lid 104. In an embodiment, lid 104 may be lined with areflective material. In an embodiment, a rigid framing (e.g., metalframing) may be added to solidify the vessel.

Vessel 102 may comprise a hole for a gas inlet 105. In an embodiment, aportion of the gas inlet inside the vessel is capped with an air stoneor metal foam 107. Additionally, vessel 102 may comprise gas vents 106to permit exit of gas from the vessel.

The vessel 102 may be of any convenient shape, for example substantiallyspherical or cylindrical. Vessel 102 may be made of food grade or highlyinert materials that do not leech and are corrosion resistant including,for example, plastics such as high-density polyethylene, low-densitypolyethylene and polypropylene. Alternatively, vessel 102 may beconstructed of stainless steel, glass and the like. It is preferred thatthe vessel be constructed of a heat resistant material and/or a materialthat can withstand light pressurization.

A lamp assembly 108 (e.g. pebble) comprising light source 109, barrier110, and an electrical connector 111 is suspended in the interior vesselvolume 112. In an embodiment, the portion of the electrical connector111 that resides inside the vessel is waterproofed. In one embodiment,the light source comprises a plurality of circuit boards 113, eachcomprising at least three edges, at least 4 edges or at least 5 edges,arranged in a substantially spherical shape defining an interior lightsource volume 114, wherein the plurality of circuit boards comprise afirst planar surface 115 in contact with the interior light sourcevolume and an opposing second planar surface 116 comprising highintensity lamps such as light emitting diodes (LEDs) 117 with anemission spectrum suitable for the growth of a photosynthetic organism;and a barrier 110 that surrounds the plurality of circuit boards formingthe substantially spherical shape. Alternatively, a single circuit board(e.g., a flexible board) may be molded into a substantially sphericalshape and used in the light assembly.

The circuit boards 116 may comprise red, white, and blue LEDs. Such LEDsmay be positioned in groups containing two or more LEDs of the samecolor positioned adjacent to two or more LEDs of another color.Alternatively, an LED including, for example, a red, white, or blue LED,may be positioned adjacent to an LED of opposing color. In someembodiments, the LEDs may be positioned in rows such that a single LEDis adjacent to four or six LEDs. The LEDs may emit light of uniformintensity or of varying intensity.

The substantially spherical shaped arrangement of the planar circuitboards may have an opening or a side devoid of at least one circuitboard to permit electrical connectivity.

The circuit board may be of any polygonal shape. In one embodiment, thepolygonal shape permits several circuit boards to be joined into asubstantially spherical shape with an interior volume. In someembodiments, the circuit boards may be pentagon shaped. In a furtherembodiment, eleven pentagons are joined to form a dodecahedron devoid ofone side (FIG. 2A). Alternatively, in some embodiments, the circuitboards may be triangular in shape. In a further embodiment, twentytriangles are joined to form an icosahedron devoid of one side (FIG.2B). In another embodiment, six triangles are joined to form a doublepyramid with a triangular base (FIG. 2C). In another embodiment, eighttriangles are joined to form a double pyramid with a square base (FIG.2D).

The circuit boards may also comprise a copper or other metal layer thatfaces the interior volume for dissipation of heat from the lamps (e.g.,LEDs) or signaling.

In some embodiments, the circuit boards comprise two or more tabs aroundtheir perimeter that form one or more notches that permit the circuitboards to interlock (see for example FIG. 3A (triangle) and FIG. 3B(pentagon).

The barrier 110 surrounding the plurality of circuit boards forming thesubstantially spherical shape may be constructed of a variety of inertmaterials, such as various plastic materials including, for example,transparent plastic and/or plastic tolerant to extreme temperatures. Thebarrier functions to provide a water-tight seal around the plurality ofcircuit boards to prevent a culture medium in the interior vessel volume112 coming in contact with the plurality of circuit boards 116. In someembodiments, the barrier 110 surrounding the plurality of circuit boardsforms a container (e.g., a jar) around the circuit boards which may besubstantially cylindrical (FIG. 3A) or spherical (FIG. 3B) in shape. Insome embodiments, the container formed by the barrier holds one or moreobjects to reduce the buoyancy of the light source (e.g., inert beadssuch as glass beads 119).

The circuit board may be populated with LEDs that emit light of one ormore wavelengths to optimize for a particular strain of photosyntheticlife or to express a specific feature for abnormal growth. In someembodiments, the circuit boards may be populated with lamps that emitelectromagnetic radiation outside the visible spectrum including, forexample, UV and/or IR light. Such lamps may be used to sterilize theliquid media in the vessel (e.g., lamps emitting UV light) and/or heatthe liquid media in the vessel (e.g., lamps emitting IR light). In someembodiments, the LEDs may be pulse width modulated. In one embodiment,the pulse width modulation is optimized to maximize growth of theparticular microorganism in the photobioreactor. In another embodiment,the duty cycle is at least 50%, at least 55%, at least 50%, at least55%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, or at least 95%.

The circuit board may comprise sensors for collecting data from theinterior of the vessel. For example, sensors may be configured tocollect data on the culture media including, for example, opticaldensity, pH, and/or conductivity.

The plurality of circuit boards forming a substantially spherical shapewith an interior volume may comprise one or more microprocessors in theinterior volume including for individual control of LEDs or control ofbanks of LEDS (e.g., two or more LEDs) and/or feedback monitoring.

Vessel 102 may comprise one or more holes or access ports for providingelectrical connectivity to the lamp assembly 108.

One or more lamp assemblies 108 may be distributed throughout theinterior vessel volume 112. A single light source may be suspended atthe center of the interior vessel volume. Alternatively, when two ormore lamp assemblies are used in the photobioreactor, the lampassemblies may be suspended at the same or different heights in theinterior vessel volume (see FIGS. 1 and 4). Alternatively, when three ormore lamp assemblies are used, the lamp assemblies may be suspended inany geometric arrangement such as a helical or double helicalarrangement.

A cooling device may be provided to control the temperature of thevessel. Such cooling device may be in the form of a cooling jacketsurrounding a portion of the wall of vessel 102. Such cooling jacketprovides circulating cooling water or other fluid across the wall ofvessel 102 to absorb heat and assist in controlling the temperature ofculture medium 103 contained in the vessel. Cooling water may enterjacket through an inlet tube and exit through an exit tube. Thedimensions of the cooling jacket will depend upon a number of factors,such as the amount of heat transmitted by lamp assemblies 108 to theliquid culture medium contained in vessel 102, the desired temperatureof the culture medium, the temperature and flow rate of the coolingwater, and the like. Alternatively, temperature of the vessel may beregulated via temperature regulation of the lamp assemblies or LEDs, forexample by circulating and/or controlling temperature of an oil in whichthe LEDs or lamp assembly is immersed.

Vessel 102 is generally designed to accommodate a head space between theliquid culture surface and lid. The head space allows for foaming, whichoften occurs in biological culture media.

Vessel 102 may be fitted with gas inlet tube 105 which is provided witha pressurized gas (e.g., carbon dioxide or carbon dioxide-enriched air)for supporting the photosynthetic requirements of the algal culture.Tube 105 passes through the wall of vessel 102. Gas bubbles rise throughthe liquid algal culture medium contained in vessel 102 and the spentgases escape through gas vents 106 which may be disposed in the wall orlid of vessel, preferably above the surface level of the culture medium.

If more vigorous mixing is desired, an air pump 118 or other agitationmechanism, for example powered by a motor, may be used to agitate theculture medium.

The photobioreactor 101 may further comprise a cleaning unit mountedwithin the interior vessel volume or on the outside surface of the lampassembly 108 for cleaning the outer surface of the lamp assembly, and acleaning unit actuator for actuating the cleaning unit. Such a cleaningunit may function to get rid of the cultivated and/or propagatedphotosynthetic organisms which may block the light source by adhering tothe outer lamp assembly. In some embodiments, the vessel is composed ofor coated with a superhydrophobic, hydrophilic, and/and or oleophobicmaterial.

Referring now to FIG. 5, this figure shows another embodiment of aphotobioreactor as provided by the instant disclosure. Such aphotobioreactor may comprise a cylindrical shaped vessel 501 (e.g., abarrel shaped vessel) to permit stacking or racking of the two or morevessels in a vertical or horizontal position. Because of their generalavailability, cost, and ease of storage (e.g., stackability), 55 gallonplastic drums can be employed with the methods disclosed herein. In anembodiment, the vessel 501 may be lined with a reflective material. Inan embodiment, the vessel comprises a lid 502 with gas exhaust fittings503, a CO₂ injection line 504, and electrical inlet fittings 505. In anembodiment, the lid may be attached to the vessel via an attachmentmechanism such as a clamp 506. The electrical inlet fittings 505 mayreceive waterproofed electrical lines 507 powered by power source 508.In some embodiments, the vessel may comprise air line bulkhead fittings509 and water line bulkhead fittings 510.

Although the embodiment shown in FIG. 5 is depicted in a verticalconfiguration, a photobioreactor consistent with the instant disclosuremay also be arranged in a horizontal configuration. Such aphotobioreactor may comprise a cylindrical shaped vessel (e.g., a barrelshaped vessel) to permit stacking or racking of the two or more vesselsin a horizontal position. In one embodiment, the vessel comprises a lidfor introducing medium or removing culture and a drain for introducingmedium or removing culture. In another embodiment, the photobioreactorcomprises a gas inlet positioned on the bottom of the photobioreactor.In one embodiment, this gas inlet is a CO₂ inlet. In another embodiment,the photobioreactor comprises one to a plurality (e.g. 1 to about 50, 2to about 30, 3 to about 20 or about 6, 8, 10 or 12) bottom gas fittingsand one to a plurality (e.g. 1 to about 50, 2 to about 30, 3 to about 20or about 6, 8, 10 or 12) of top gas fittings. In one embodiment, asviewed from the base end of the vessel with the drain at the bottom, thebottom gas fittings are located substantially between about the 3- andabout the 6-o'clock positions, for example about the 3-, about the3:30-, about the 4-, about the 4:30-, about the 5-, about the 5:30- orabout the 6-o'clock positions. In one embodiment, as viewed from thebase end of the vessel with the drain at the bottom, the top gasfittings are located substantially between about the 9- and the12-o'clock positions, for example about the 9-, about the 9:30-, aboutthe 10-, about the 10:30-, about the 11-, about the 11:30- or about the12-o'clock positions. In one embodiment, air is circulated in and out ofthe vessel via the bottom gas fittings and the top gas fittings. In oneembodiment, gas enters the vessel via the bottom gas fittings and exitsthe vessel via to gas fittings. In one embodiment, the vessel containsvolume markers, for example gallon markers.

The photobioreactor may further comprise a transferring mechanism fortransferring liquid media, nutrients and/or antibacterial agents to theinterior vessel volume. Nutrients may include synthetic and/or organicnutrients. In some embodiments, an anti-bacterial agent (e.g., adetergent) may be added to the liquid media to slow, or prevent, thegrowth of contagens (e.g., any organism that is other than thephotosynthetic microorganism purposefully added to the vessel) in thevessel. Nutrients and/or antibacterial agents may be added to the vesselby any method known in the art including, for example, via an automateddrip system. In an embodiment, the automated drip system may beconnected (e.g., wired or wireless connection) to a monitoring systemthat monitors nutrient levels in the vessel. Such a monitoring systemmay constantly or periodically monitor nutrient levels in the vessel andprompt the automated drip system to release nutrients into the vesselwhen nutrient levels fall below a predetermined limit. Conversely, themonitoring system may prompt the automated drip system to stop therelease of nutrients into the vessel when nutrient levels exceed apredetermined limit. In some embodiments, other trace chemicals (e.g.,citric acid) may be added to the vessel to optimize environmentalconditions for the growth of the photosynthetic microorganism.

FIGS. 6A to 6F show embodiments of lamp assemblies 108 included within avessel 102. In each of these embodiments, the lamp assemblies 108 areconfigured to be interconnected in series along one or more connectionlines 602. This connection scheme enables power to be provided to morethan one lamp assembly 108 within a vessel. The configurations of thelight assemblies 108 within the vessels 102 are based on the strain inquestion and the targeted product. For example, certain strains may havebetter growth rates when only one connection line 602 is disposed withina vessel while other strains have better growth rates when more than oneconnection line is disposed within a vessel.

While FIGS. 6A to 6F show the connections being made in series, the lampassemblies 108 may include parallel electrical connections so that afailure of one lamp assembly does not affect the operation of otherinterconnected lamp assemblies. Further, while the connection lines 602are shown as being substantially straight through the vessels 102, inother embodiments, the connection lines 602 may be formed at one or moreangles (e.g., lamp assemblies 108 may be connected at a 90 degreeangle).

The example connection line 602 is configured to provide at least one ofpower and immersion oil to each of the lamp assemblies 108. For example,in FIG. 6A the vessel 102 a includes connection line 602 a, whichincludes ports 604 a and 604 b. The immersion oil is provided to theconnection line 602 a through port 604 a and exits the connection line602 a at port 604 b. In this manner, an operator can cycle immersion oilthrough the lamp assemblies 108 connected to the connection line 602 ato control temperature. In addition, electrical connector 111 isprovided to each of the lamp assemblies 108 through port 604 a. Thecontrol of power and oil to the lamp assemblies 108 is described infurther detail in conjunction with FIGS. 9 and 10.

FIG. 6C shows that connection lines 602 may interconnect. Thisinterconnection facilitates the flow of immersion oil through the vessel102. Additionally or alternatively, the interconnection may enable lampassemblies 108 located at intersection points to be controlled by eitherof the control signals on the intersecting electrical connectors 111.For instance, the lamp assembly 108 c may be connected to electricalconnectors 111 positioned within respective ports 604 c, 604 d, and 604e. As a result, the lamp assembly 108 c may be controlled by a controlsignal provided on any one of the electrical connectors 111.

FIG. 6D shows a connection line 602 with a lamp assembly 108 thatincludes twenty circuit boards with LEDs. In this embodiment, immersionoil is cycled though the lamp assembly 108 to control temperature.Further, the configuration of the twenty circuit boards enables light tobe directed to substantially any location within the vessel 102. This isin comparison to the light assemblies 108 of FIGS. 6A, 6B, 6C, 6E, and6F that include relatively fewer circuit boards. These figuresaccordingly use more light assemblies to compensate for each lightassembly having fewer LEDs.

A large scale photobioreactor is also contemplated by the presentdisclosure. Such a bioreactor may comprise one or more vessels and one,two or more lamp assemblies. The vessels may be of uniform volume anddimension or may be of varying volume and dimension. For example,vessels that hold 55 gallons or greater, or vessels that hold 3,000gallons or greater, may be used in the large scale photobioreactorsdisclosed herein. In an embodiment, area may be provided between vesselsto provide for connectivity of one vessel to another. In anotherembodiment, the vessel may have a central column for light distributionand/or electrical connectivity.

It will be appreciated that the photobioreactors of the invention can beproduced in widely varying sizes. Several photobioreactors may begrouped together to produce large scale photobioreactors for example asshown in FIGS. 7 and 8. For simplicity, a single photobioreactoremploying one light assembly has been illustrated in FIG. 1, however, ina typical industrial scale photobioreactor, 2, 3, 4, 5, 6 ,7, 8, 9, 10,20, 30, 40, 50, 60, 70, 80, 90, 100 or more photobioreactors eachcomprising one or more lamp assemblies may be used.

A photobioreactor with two or more vessels may comprise air and waterlines that connect a vessel with one or more adjacent vessels. The airand water lines may comprise valves to direct the flow of air and/orwater in a predetermined manner. Optionally, the valves may permit forquick disconnection of a vessel for maintenance and/or inspection. FIGS.7 and 8 show diagrams of photobioreactor embodiments including multipleinterconnected vessels. In particular, FIG. 7 shows a photobioreactorembodiment with multiple columns of vessels wherein each column ofvessels is interconnected to another column of vessels. FIG. 8 shows aphotobioreactor embodiment wherein multiple vessels in a single columnare interconnected.

In the illustrated embodiment of FIGS. 7 and 8, air enters in throughthe twelve gas inlets at the 7:30 and 4:30 positions on the barrel wherethe stream is broken up into a column of two micron bubbles, which mixwith the culture medium, adding circulation and necessary gases. Forstrains with lower gas requirements it is possible to subdivide the gasinlets using a fraction of the inlets for gas and leaving the remainderof the inlets unused or connected to a water recirculation pump. In thisembodiment, a gas bubble builds at the top of the lower barrels untilthe gas reaches the 11:30 and 12:30 positions of the gas outlet. At thispoint, the pressure of the bubble drives the same process in the barrelabove until the gas reaches the top of the column. It should also benoted that the bottom barrel of each column includes between two andfour additional gas inlets coupled to 0.5 micron diffusers, which allowfor the utilization of more exotic gases for gross supplementation (i.e.CO₂, gaseous ammonia, etc.).

In some embodiments, a large scale photobioreactor may comprise a singleinjection point for air and/or nutrients such that the introduction ofair (e.g., CO₂) and/or nutrients into a first vessel operably connectedwith other vessels allows the air and/or nutrients to move from thefirst vessel into the other vessels.

The vessels of a large scale photobioreactor may be operably connectedto a control unit (e.g., a controller or a server) and/or sensormonitoring unit. In some embodiments, the control unit may be configuredto permit an operator to control the lamp assembly in each vessel (e.g.,turn the lamp assembly on or off, or adjust the intensity of the lamps).In some embodiments, the sensor unit may be configured to disconnect avessel from other vessels in the photobioreactor upon sensing that thevessel has been contaminated.

A high level block diagram of an example control system 900 isillustrated in FIG. 9. The example control system 900 includes aworkstation 902, a controller 904 (e.g., a control unit), a vessel 102,pumps 906, and an immersion oil container 908. While FIG. 9 shows thecontroller 904 providing control for only the vessel 102, in otherembodiments the controller 904 may be communicatively coupled to two ormore vessels. Further, the workstation 902 may be communicativelycoupled to more than one controller.

The example workstation 902 is configured to provide an operatorinterface for providing instructions to the controller 904, controllingof the process, and displaying system process data. The examplecontroller 904 manages the control of lamp assemblies 108 and the flowof immersion oil. In other examples, the controller 904 can beconfigured to manage the control of the liquid culture medium 103 withinthe vessel 102. For instance, the controller 904 may be configured tocontrol gas inlet valves, gas vents, fluid inlet valves, and/or additivevalves.

The example workstation 902 operates in conjunction with the controller904 to provide control and system feedback to an operator. Theworkstation 902 includes any type of processor including, for example, apersonal computer, a laptop, a server, a smartphone, a tablet computer,etc. The controller 904 includes any type of control system (e.g., anArduino™ microcontroller or an Application Specific Integrated Circuit(“ASIC”)) that is configured to provide open or closed loop systemcontrol using inputs from sensors to control lighting and oil flow.

In this example, the controller 904 is separately electrically connectedto lamp assemblies 108 a-d via respective electrical connectors 111 aand 111 b. The electrical connectors 111 are disposed within respectiveconnection lines 602 a and 602 b. The controller 904 uses the separateconnection lines 602 to provide separate control to the lamp assemblies108 within the respective connection lines 602. For example, thecontroller 904 may provide power to the lamp assemblies 108 a and 108 bwhile placing lamp assemblies 108 c and 108 d into an off state.

The example controller 904 is communicatively coupled to the pumps 906.The controller 906 may provide digital instructions or an analog PWMvoltage to operate the pumps 906. In this example, the system 900includes a separate pump 906 for each of the connection lines 602 sothat the controller 904 can independently control the flow of immersionoil to the respective lamp assemblies 108. In other embodiments, asingle pump 906 is used to provide oil to one or more connection lines602.

The example pumps 906 include any type of component for providingimmersion oil to the connection lines 602. For example, the pumps 906can include displacement pumps, gear pumps, screw pumps, roots-typepumps, peristaltic pumps, plunger pumps, hydraulic pumps, velocitypumps, etc. Responsive to receiving a control signal (e.g., a voltage)from the controller 904, the pumps 906 move immersion oil from thecontainer 908 to the respective connection lines 602. The pumps 906 canbe configured to pump oil at varying velocities. Alternatively, thepumps 906 may be configured to operate in a binary state (e.g., On/Off).While the pumps 906 are shown as being located at the entrance of theconnection lines 602, in other embodiments the pumps 906 may be locatedat the exit of the connection lines 602. In these other embodiments, thepumps 906 are configured to pull immersion oil through the connectionlines 602.

The example immersion oil container 908 may be any type of tank to storeimmersion oil. In some examples, the container 908 may include a jacketthat is positioned along a portion of the vessel 102. Further, the oilcontainer 908 may include a component that provides active cooling orheating.

In the illustrated example of FIG. 9, the controller 904 is configuredto control a voltage, frequency, and PWM (e.g., duty cycle) of powerapplied to the lamp assemblies 108. For example, the controller 904 isconfigured to provide a voltage between 8.8 and 12 volts to each of thelamp assemblies 108. In other examples, the controller 904 may beconfigured to provide a voltage between 0 and 24 volts to the lampassemblies 108. The applied voltage is used to control the intensity oflight transmitted from the lamp assemblies 108. In examples where theLEDs (or other light sources) emit light proportional to the appliedvoltage, the controller 904 is configured to provide a voltage such thatthe LEDs emit light at a light intensity optimal for growth of thecurrent culture.

The frequency of light emitted by the LEDs may be in the range between390 to 700 nm. The frequency of light is based on the type of lightsource. In some examples, the controller 904 is configured to selectwhich LEDs are activated to achieve the desired light frequency. Forinstance, each of the lamp assemblies 108 may include red, green, andblue LEDs. The lamp assemblies 108 may also include one or more mirrorsto combine the light transmitted from the LEDs. The controller 904 maybe configured to time the pulsing of the different colored LEDs toachieve a resulting light frequency that is optimal for the targetculture. Alternatively, light filters may be applied to the lampassemblies 108 to achieve the desired light frequency.

In addition to controlling the magnitude of the voltage, the controller904 is also configured to cycle the voltage at a specified PWM. Forexample, the controller 904 may cycle the lamp assemblies 108 over a 100millisecond (ms) time period such that the LEDs are on for 75% of thetime (e.g., 75 ms) and off for 25% of the time. The controller 904 isaccordingly programmed (or provided instructions) for a duty cycle andtime period. While a 75% duty cycle and 100 ms time period was used asan example, the controller 904 can be programmed to operate the lampassemblies 108 using any duty cycle and time period.

The controller 904 is also configured to control the flow of immersionoil through the lamp assemblies 108 to regulate the temperature withinthe vessel 102. The controller 904 uses instructions provided from theworkstation 902 to determine how the power and oil flow is to becontrolled. It should be appreciated that different types of cultureshave optimal growth settings and as a result, the controller 904 can beprogrammed based on the culture to be grown within the vessel 102.

The example controller 904 is also configured to monitor conditionswithin the vessel 102 and report these conditions to the workstation902. In this embodiment, the vessel 102 includes temperature sensors912, which are communicatively coupled to the controller 904. Thecontroller 904 records temperature data provided by the sensors 912 andperiodically transmits the temperatures to the workstation 902. Thecontroller 904 is also configured to report current operating conditionsincluding, for example, voltage, frequency, and PWM applied to each lampassembly, a duration of operation, and/or detected diagnostic faultswithin the system 900 (e.g., a broken lamp assembly or obstructionwithin a connection line 602), communication interference with theworkstation 902, etc.

FIG. 10 shows a flow diagram illustrating example procedures 1000 and1050 to manage the growth of a culture within the vessel 102 of FIG. 9,according to an example embodiment of the present invention. The exampleprocedures 1000 and 1050 may be carried out by, for example, theworkstation 902, the controller 904, the pumps 906, and/or the lampassemblies 108 described in conjunction with FIG. 9. Although theprocedures 1000 and 1050 are described with reference to the flowdiagram illustrated in FIG. 10, it will be appreciated that many othermethods of performing the acts associated with the procedures 1000 and1050 may be used. For example, the order of many of the blocks may bechanged, certain blocks may be combined with other blocks, and many ofthe blocks described may be optional.

It will be appreciated that all of the disclosed procedures describedherein can be implemented using one or more computer programs orcomponents. These components may be provided as a series of computerinstructions on any conventional computer-readable medium, includingRAM, ROM, flash memory, magnetic or optical disks, optical memory, orother storage media. The instructions may be configured to be executedby a processor (e.g., the workstation 902 and/or the controller 904),which when executing the series of computer instructions performs orfacilitates the performance of all or part of the disclosed methods andprocedures.

The example procedure 1000 begins when an operator uses the workstation902 to program a routine for a session to grow a new culture (e.g., aphotosynthetic algae culture) (block 1002). The operator specifies atime period of 50 ms and a duty cycle of 55%. The operator alsospecifies that the voltage applied to the LEDs is to be 10.75 volts togenerate a desired light intensity. The operator further specifies thatthe culture is to be grown over a two day period and that thetemperature of the medium 103 is not to exceed 105° C. The operatormoreover selects to receive alerts when any issues are detected with anyof the lamp assemblies 108 and a report that graphs the temperature ofthe vessel 102 over time.

After programming the routine, the operator instructs the workstation902 to transmit instructions 1003 (e.g., parameters) to the controller904 (block 1004). The instructions 1003 include the programmed routineand may be formatted in a programming language compatible with thecontroller 904. The workstation 902 then begins to receive and storeprocess data 1005 from the controller 904 (block 1006). The process data1005 includes operational and diagnostic information indicative of theprocess at the vessel 102. The workstation 902 may receive a relativelyconstant stream of process data 1005 as the data is processed andtransmitted by the controller 904. Alternatively, the workstation 902may periodically receive the process data 1005 from the controller 904.

The workstation 902 analyzes the process data 1005 for any alerts (block1008). Additionally or alternatively, the workstation 902 determineswhether an alert message 1007 was received from the controller 904. Ifan alert was received, the workstation 902 notifies an operator of thealert (block 1010). The notification can include, for example, a textmessage, an e-mail, an audio message, etc. indicating the contents ofthe alert. In the illustrated example, an alert can include anindication that a temperature of the vessel 102 is greater than the 105°C. threshold. Alternatively, the alert can include informationindicating that one or more of the lamp assemblies are inoperable.Moreover, the alert can include information indicating that either ofthe pumps 906 is not performing as expected.

In the illustrated example of FIG. 10, the workstation 902 determineswhether the session has expired after providing an alert to the operator(block 1012). In other embodiments, the workstation 902 may instruct thecontroller 904 to pause the process until an operator responds to thealert. In yet other embodiments, the workstation 902 may provide thecontroller 904 instructions for responding to an alert. For instance,after detecting that the lamp assembly 108 b is inoperative, theworkstation 902 provides the controller 904 instructions changing theduty cycle, time period, and voltage for the other lamp assemblies 108a, 108 c, and 108 d to compensate for the loss of light.

Responsive to not receiving an alert, the workstation 902 determineswhether the session (e.g., the programmed two day operating time) hasended (block 1012). Responsive to determining the session has ended, theworkstation 902 receives a report 1013 that includes the monitoredtemperature of the vessel 102 over the two day period (block 1014). Theexample procedure 1000 then ends. Alternatively, the example procedure1000 returns to block 1002 for the next session. However, if theworkstation 902 determines that the session has not ended (block 1012),the workstation 902 returns to receiving process data 1005 from thecontroller (block 1006). The example procedure 1000 then continues untilthe session expires.

The example procedure 1050 begins when the controller 904 receivesoperation instructions 1003 from the workstation 902 (block 1052). Theexample controller 904 then configures its operation to provide power tolamp assemblies 108 based on the instructions 1003 (block 1054). Theconfiguration can include setting parameters for output drives so thatthe lamp assemblies 108 receive 10.75 volts at a 55% duty cycle of a 50ms time period. The example controller 904 also configures itsconnection to the pumps 906. This configuration can include operatingthe pumps 906 so that there is sufficient immersion oil within the lampassemblies 108.

The example controller 904 also configures operation conditionsincluding, for example, an allowable temperature ranges for the vessel102, alert threshold temperatures for the vessel 102, diagnosticsettings, etc. (block 1056). The controller 904 also determines whichprocess data is to be transmitted to the workstation 902 and which datais to be included within a report. The configuration can further includecalibration of the temperature sensors 912 and/or the lamp assemblies108.

After configuration, the controller 904 operates the lamp assemblies atthe specified voltage and duty cycle (block 1058). The examplecontroller 904 also begins receiving outputs from the temperaturesensors 912. For instance, temperature sensor 912 a reports thetemperature in the liquid medium culture 103, temperature sensor 912 breports the temperature within the lamp assembly 108 a, and temperaturesensor 912 c reports the temperature of connection line 602 b. It shouldbe appreciated that in other examples, only one of the temperaturesensors 912 may be used. It should also be appreciated that in otherexamples, the controller 904 may be configured to receive outputs fromother types of sensors (e.g., pH sensors, salinity sensors, lightsensors, chemical sensors, etc.

In this example, the controller 904 transmits the temperature data asthe process data (e.g., operational data) 1005 (block 1060). The processdata 1005 can also include any faults detected within the vessel 102and/or within the controller 904. The process data 1005 can furtherinclude the voltage and duty cycle applied to the lamp assemblies 108and whether the pumps 906 are being operated.

The example controller 904 then determines whether the session shouldend (e.g., the two day period) (block 1062). Responsive to determiningthat the session should end, the example controller 904 compilescollected data, generates a report, and transmits the report 1013 to theworkstation 902 (block 1064). In other embodiments, the examplecontroller 904 ends the session and notifies the workstation 902 thatthe session has ended without providing a report. In these otherembodiments, the controller 904 may not have the capability ofgenerating a report. Instead, the controller 904 may provide a log ordata structure of collected data at the end of the session. At the endof the session, the example procedure 1050 ends. Alternatively, theexample procedure 1050 returns to block 1052 to begin a session foranother culture.

Returning to block 1062, if the session has not ended, the controller904 compares the temperature outputs from the sensors 912 topre-specified temperatures (block 1066). The controller 904 performsthis comparison to determine whether action should be taken to activelychange the temperature within the vessel 102. For example, if thetemperature is outside of a specified allowable range, the controller904 operates the pumps 906 to circulate cooler (or warmer) oil throughthe vessel 102 (block 1068). The controller 904 continues to operate thepumps 906 until the temperature is within the allowable range.Additionally or alternatively, the controller 904 may also change theintensity of the lamp assemblies 108 and/or the duty cycle to modify thetemperature. For instance, lowering the voltage applied to the lampassemblies 108 reduces the amount of heat transmitted to the vessel 102.Moreover, changing the duty cycle to have more time in an ‘Off’ statealso reduces the amount of heat transmitted to the vessel 102.

The example controller 904 also determines whether the temperature ofthe vessel 102 exceeds a threshold (block 1070). Responsive todetermining the temperature exceeds a threshold, the controller 904transmits an alert message 1007 to the workstation 902 (block 1072). Thecontroller 904 continues to operate the lamp assemblies 108 untilfurther instruction is provided by the workstation 102 or until thesession ends.

A photosynthetic microorganism such as an alga may be cultivated and/orpropagated with the photobioreactor as disclosed herein. Generally,prior to filling the interior volume of the vessel of thephotobioreactor with a culture medium, the interior of thephotobioreactor is sanitized by exposing it to a sterilizing gas, ahypochlorite solution or the like. Following sanitization, water isintroduced into the vessel via a pressurized water line. The vessel isfilled with water to a predetermined depth. In some embodiments, thevessel is filed such that the surface of the culture medium will bebelow an exit port. Nutrients and inocula (e.g., one or morephotosynthetic organisms such as an alga) are introduced into the vesselby removing the lid or introducing them through an access port in thevessel or lid. The pH and temperature of the medium may be monitoredthroughout the photobioreaction by a temperature probe and a pH probe.The photobioreaction is initiated by supplying electrical power to thelamp assembly and optionally initiating sparging of an appropriate gasmixture via an inlet tube. The progress of the photobioreaction may bemonitored with a calibrated density detector. Adjustments to the pH orthe composition of the culture medium may be effected by introducingmaterials through the lid or an access port in the vessel or lid.

In some embodiments, a starter culture of each photosyntheticmicroorganism to be added to the vessel may be grown. After the starterculture reaches an optimal density, a portion of the culture may beadded to the vessel. In some embodiments, where two or more types (e.g.,species or strains) of microorganisms are added to the vessel, an equalnumber of each type of photosynthetic microorganism may be added to thevessel. Alternatively, an unequal number of each strain ofphotosynthetic microorganism may be added to the vessel.

When the photosynthetic microorganisms are ready to harvest, the lightsource and optional sparging gas are turned off and the mediumcontaining the photosynthetic microorganisms is collected. In anembodiment, the medium and photosynthetic microorganism may be collectedvia the opening of an optional valve located on vessel wall. In afurther embodiment, a pump may be used to expel the medium andphotosynthetic microorganism from the vessel. Alternatively, inembodiments where the vessel comprises a gas inlet (e.g., for CO₂injection), the gas inlet may be reversed to allow static pressure tobuild up and force the media out via an alternate line. Alternatively,the photosynthetic microorganism may agglomerate and be expelled fromthe vessel (e.g., by excess air flow) onto a surface external to andapart from the vessel. In other embodiments, an ultrasonic transducermay be used to vaporize the media in the vessel and thereby cause a massof photosynthetic microorganisms to collect around the transducer wherethey may subsequently be harvested. Optionally, the vaporized media maybe collected and recirculated for use in the photobioreactor. Themicroorganisms may then be harvested and/or dehydrated by any methodsknown in the art.

The photobioreactor may further comprise a separating apparatus forseparating the removed portion of media containing the photosyntheticmicroorganisms into a liquid phase and into a solid phase (whichcontains the microorganisms). The separating apparatus is preferably afilter but depending on the type of microorganisms, other separatingmeans known to one skilled in the art may be used.

In an exemplary method, the photobioreactor disclosed herein may be usedto propagate one or more photosynthetic microorganisms (e.g., apolyculture) such as algae that produce biomolecules such as fattyacids, phycobiliproteins such as C-Phycocyanin, allophycocyanin,phycoerythrin, biofuels such as phytol, and other various petrol fuelsubstitutes. In one embodiment, two or more algae may be propagatedtogether that have natural environments that are similar in salinity anddissimilar in temperature such as algae selected from the groupconsisting of isochrysis aff. galbana, pavlova lutheri, arthrospiraplatensis, chlorella pyrenoidosa, synechococcus elongates, includingnaturally occurring or genetically modified/recombinant strains of theforegoing.

Such a combination of algae provides the benefit that while thetemperature of the liquid media in the vessel may change, there willtypically be at least one alga that propagates at a low temperature andat least one alga that propagates at a higher temperature. Algae arethen propagated in the photobioreactor as provided herein and ultimatelyseparated from the growth medium after the algae reach a desireddensity. After separation of the algae from the growth media, DHA isobtained from the algae and optionally purified.

Without further description, it is believed that one of ordinary skillin the art may, using the preceding description and the followingillustrative examples, make and utilize the agents of the presentdisclosure and practice the claimed methods. The following workingexamples are provided to facilitate the practice of the presentdisclosure, and are not to be construed as limiting in any way theremainder of the disclosure.

EXAMPLES Example 1 Construction of Pebble

A pebble (also referred to herein as lamp assembly), for practice of themethods provided herein, may be constructed by any known methods andmaterials in the art and comprises a light source and a barriersurrounding the light source.

A. Pebble Assembly

A pebble may be constructed from a circuit panel (e.g., pentagonalshaped panels) with pre-mounted LED array. A pebble section (e.g., twoor more affixed pentagon-shaped circuit boards) is prepared by bondingeach with liquid cyanoacrylate (1 three-panel section, 2 four-panelsections) along internal edges, not connecting one section to the other.The cyanoacrylate is given approximately 3 hours to dry. After drying,three sections are press fit together into a dodecahedral shape, withone side of the dodecahedron kept open for connectivity purposes (e.g.,wire access), and allowed to dry and settle into shape overnight. Next,the three main sections are taken apart. On each of the two 4-sidedpebble sections, using lead-free solder only, ground connections with 28gauge wire are soldered to the central panel. This is repeated with a12-volt connection, making connection to same central panel as groundand with 3-sided portion, using any of the 3 sides as the mainconnection panel. The ground connection from the main panel on the3-panel section is then connected to the main panel on one of the4-panel sections with 28 gauge wire and lead-free solder. The second4-panel section's main panel is connected to the first 4-panel sectionon the panel on the far end on the section adjacent to the main panel(sharing a side with the main panel, with only one other side bonded toa panel). To this last panel, now connecting all the ground on onecircuit and all the 12V connection on another, solder a 22-gauge wire toeach circuit (length to be determined by even pebble distributionthroughout barrel (approximately 10″-40″)). Next, the panels are pressfit together, taking care to carefully arrange wires within. The pebblesare then filled with 10-12 glass beads for weight.

B. Pebble Case Assembly

A case for the pebble may be prepared using a polyethylene (Nalgene) jarwith dimensions to allow for the circumference of the pebble. A ¼″ holeis drilled in the center of the lid of the jar. Next, a silicone gasketis inserted ¼″ through the wall of the lid. A male ¼″ brass through-wallbarbed fitting is then screwed from underside of lid and attached to a¼″ silicone gasket from top of the lid. A female ¼41 brass fitting isthen screwed to the male fitting from the top of the lid. Next, 1½″ of¼″ inner diameter flexible silicone tubing is attached to the lid gasketusing cyanoacrylate as a bonding agent. ¼″ OD rigid LLDPE white tubing(length to be determined by optimal pebble dispersion within barrel) isthen attached to connection tubing using cyanoacrylate as a bondingagent. The base of a jar is then filled with a number of clear glassbeads (e.g., 14) for weight and light dispersion. The assembled pebbleis then placed within the jar and seated upon glass beads. 22 gauge wireis then threaded through the gasket and tubing. Subsequently, the jar isfilled to maximum with white mineral oil and the lid is tightly screwedon expelling as much air as possible. Excess oil adhered to the outsideof the jar is removed and the jar is then rinsed with reverse osmosiswater.

C. Pebble Case Embodiments

FIGS. 12 to 35 are diagrams showing embodiments of lamp assemblies. Ineach of the embodiments, the lamp assemblies 108 include circuit boards113 connected together in a specific geometry based on the dimensions ofthe casing. Each of the lamp assemblies includes an inlet and an outletto facilitate the flow of immersion oil. The inlet and outlet aredimensioned to mechanically connect to the connection lines 602.Further, the inlet includes electrical connectors 111 (which are notshown).

For example, FIGS. 12 and 13 show a lamp assembly 108 that includes sixcircuit boards 113 connected together to form a double-pyramid. The lampassembly 108 includes a casing 1202 that is dimensioned to accommodatethe six circuit boards. In particular, the casing 1202 is bulb-shaped,which provides an efficient propagation of light to a surrounding liquidculture medium. The lamp assembly 108 receives immersion oil via theinlet 1204. The immersion oil exits the lamp assembly 108 at output1206. Further, electrical connectors 111 are routed through the inlet tothe circuit boards 113 (not shown). In some examples, the electricalconnectors 111 contact a first circuit board. Electrical traces are usedto electrically connect the other circuit boards to the first circuitboard. In other examples, each of the circuit boards 113 is connected tothe electrical connectors 111.

FIGS. 14 to 16 show schematic diagrams of the lamp assembly 108 of FIGS.12 and 13. In particular, FIG. 14 shows a top-perspective view of thelamp assembly and FIG. 16 shows an enlarged view of a case connection1504 of the casing 1502 shown in FIG. 15. In this embodiment, the casing1502 is formed as two separate halves joined together at joint 1600. Thecasing 1502 is formed as separate halves to enable the circuit boards tobe installed inside the casing 1502. As shown in FIGS. 15 and 16, thejoint 1600 may be mechanically sealed by the dimensioning of each halfof the casing 1502. Alternatively, the joint 1600 may be chemicallysealed using an (substantially transparent) adhesive.

FIGS. 17 to 22 show another embodiment of a lamp assembly 108. In thisembodiment, a casing 1702 is more spherical than the casing 1502. Inaddition, FIGS. 19 to 22 shows that the casing 1702 is formed of twohalves that are connected together using tabs 1902. In particular, FIGS.21 and 22 show how the halves of the casing 1702 are mechanicallyconnected by each of the tabs contacting a corresponding reception area.

FIGS. 23 to 28 show different embodiments of circuit boards within thelamp assemblies. In particular, FIG. 23 shows a top-perspective view ofa lamp assembly 108 shown in FIG. 24. The lamp assembly 108 includes atriangular matrix of circuit boards 113 connected together to form acube. The connection of the circuit boards 113 enables light to betransmitted in substantially all directions for optimal culture growth.

FIG. 25 shows a diagram of an eight-sided cube formed from triangularcircuit boards 113. FIG. 26 shows a diagram of three-sided cube formedfrom triangular circuit boards 113. FIG. 27 shows a diagram of six-sideddouble pyramid formed from triangular circuit boards 113. FIG. 28 showsa diagram of six-sided cube formed from triangular circuit boards 113.It should be appreciated that other embodiments can include greaternumber of sides to form a structure that is substantially spherical.

Example 2 Construction of Photobioreactor

A photobioreactor for practice of the methods provided herein may beconstructed by any known methods and materials in the art and isconstructed by assembly of a barrel, lid, and CO₂ stone (also referredto herein as a lamp assembly).

A. Assembly of Barrel

In an exemplary method, using a standard 60 gallon polypropylene barrel(open head), the barrel is prepared by drilling 8 equidistant ½″ holes4½″ from the base of the barrel. Equidistant between two adjacent holes,a 9th ½″ hole is drilled 2½″ from the base of the barrel. Next, using anexacto blade, excess plastic is removed and the drill hole edges aresmoothed.

From the inside of the barrel, using a wrench, a ¼″ barbed male nylonfitting is screwed through each of the drilled holes. Next, from outsidethe barrel, ½″ silicone washer fittings are then attached onto thebarbed male fittings which are then screwed onto ¼″ nylon barbed femalefittings and tightened. 1¾″ length of rigid (thick wall) ¼″ tygon tubingis then attached to the male fitting on the outside of the barrel.Subsequently, ½″ length of black ⅛″ black silicone tubing is slid overthe barbed fitting of a large air stone. The air stone assembly is theninserted into the tygon tubing and the process is repeated for all ½″fittings at the 4½″ height. A 3″ length of rigid tygon tubing (thickwall) is then attached to an external, female barbed fitting. Thisprocess is then repeated for all 9 fittings. Next, 130″ of rigid whiteLLPDE tubing is attached to all 8 fittings at the 4½″ height. Apush-to-connect adaptor fitting^(i) and push-to-connect ball valve arethen attached to the 9th (2½″ height) fitting. Subsequently, 2″ of clear¼″ tygon tubing (thin wall) is attached to each of the 130″ white rigidLLDPE tubes. 8½″ lengths of ⅛″ black silicone tubing are then connectedto barbed fittings of an 8-way polypropylene manifold. Each rigid LLPDE130″ tube is connected to the 2″ long clear tygon tubing connector (thinwall), which is then connect to the manifold.

B. Assembly of Lid

In an exemplary method, seven holes are drilled into a blackpolypropylene lid using a ½″ plastic drill bit, with a configuration asshown in FIG. 11A.

Any excess plastic is then cleaned away from the drilled edges with anexacto blade and smoothed. From the bottom of the lid, ¼″ male barbednylon fittings are placed in the holes and screwed together with ¼″female barbed nylon fittings from the top side of the lid. Siliconewashers may be placed between the female fitting and the top side of thelid. Distribution of lights using other fittings to be determined by thestrain(s) intended for cultivation. Next, 2″ length of rigid tygontubing (thick wall) is attached to each male nylon ¼″ fitting in thelid. A length of rigid LLPDE tubing is then attached to each connection,except the central location (length to be determined by evendistribution of pebbles throughout barrels) as shown in FIG. 11B.

Subsequently, 2″ rigid tygon connector tubing is then connected to anend of the rigid LLPDE tubing. The pebble wiring is run through therigid LLPDE tubing and out through the barbed fittings in the top of thelid. Connector tubing is then attached to the base of pebble's brassfitting using liquid cyanoacrylate as a binding agent. This process isrepeated as needed for desired pebble distribution. For protection fromsalt contamination, flexible latex tubing is attached to top, female endof lid fittings and around all wires as needed based on distribution ofsaltwater spray.

C. Assembly of CO₂ Stone

In an exemplary method, 30″ length of rigid white LLDPE tubing isattached to a central fitting with connector tube. Next, the dome apexis marked on the female hemisphere of a 2 part 100 mm clear acrylicsphere. The corners of an equidistant triangle, with sides approximately2″ in length, are then marked out on the male hemisphere and centered onthe dome apex. A ¼″ rotary tool grinding bit is then used to makepreliminary guide holes for each marking. Next, a ½″ rubberized grindingbit is used to make final holes centered on guide holes. Excess plasticfrom drilling is removed from the holes is removed with clippers and anexacto blade until the holes are smooth. A ½″ rubber through-wall gasketis then attached to each of the holes. Next, a ½″ brass through-wallbarbed fitting is attached to the central hole in the female hemispherewith a gasket. The male hemisphere is then filled with 450 grams ofclear glass beads. Subsequently, cyanoacrylic bonding agent is appliedto the bottom male hemisphere and the male and female hemisphere arepress fit together. The cyanoacrylic bonding agent is allowed to dry forapproximately one hour. Next, 2″ length of rigid tygon is attached to abrass fitting barb using cyanoacrylic bonding agent. Again, thecyanoacrylic bonding agent is allowed to dry for approximately one hour.The assembled sphere and connector tube are then attached to rigid whitetubing attached to central lid fitting.

Example 3 Use of Photobioreactor for Growth of PhotosyntheticMicroorganism

A photobioreactor provided herein may be used in methods for growing oneor more photosynthetic microorganisms. Such methods may employ a fourstep process including: 1.) determining optimal environmental conditions(OEC); 2.) staging and inoculation of production environment; 3.)growing a photosynthetic microorganism to an extractable mass; and 4.)selective extracting of mature cells.

A. Determination of Optimal Environmental Conditions (OEC)

When starting up a new or unknown strain it is necessary to determineoptimal values for all growth variables including: salinity, nutrientconcentrations, EM frequency (RWB ratio), EM cycle, and rate of airflow.OEC may be determined by using an approximation of natural environmentalconditions (NEC) as a starting point. Beginning with three Generation 4reactor chambers (2 liter capacity) for each variable to be tested,process of elimination is used to narrow down the options. For example,chamber 1 comprises an experimental variable at a concentration 60%greater than its NEC, chamber 2 comprises the experimental variable atits NEC, and chamber 3 comprises the experimental variable at aconcentration 60% less than its NEC. Speed of growth of the strain isdetermined by observation of the rate in change of Diffused OpticalDensity (DOD). If/then for DOD:

If C1>C2>C3, Then Round 2 baseline=C1 with variances of +/−15%;

If C2>C1&C3 , Then Round 2 baseline=C2 with variances of +/−15%;

If C3>C2>C1, Then Round 2 baseline=C3 with variances of +/−15%

This process is continued for 4 rounds and repeated for eachexperimental variable to determine the new strain's OEC values.

B. Staging and Inoculation of Production Environment

Using the OEC values determined above, a Generation 5 Reactor (30 litercapacity) is prepared to those levels determined in A above. Salinitylevels are set, pumps are activated, and nutrients are added to thereactor in accordance with such predetermined levels. The nutrients aregiven approximately two hours to mix without light. After the nutrientshave mixed, the Reactor is seeded with at least 10% live culture. Basedon continuous testing of water nutrient levels, the culture is fed asneeded for the next 3-10 days based on the growth rate of the strain.When DOD has reached a level where individual LED's are no longervisible, a generation 7 reactor is prepared using the same method asdescribed above with 90% of the culture in Generation 5 Reactor asinoculation for Generation 7 Reactor.

C. Growth to Extractable Mass

Once growth is established in the Generation 7 Reactor, the followingconditions should be continually monitored: pH, ammonia, nitrate,nitrite, phosphate, dissolved CO₂, dissolved O₂, system air flow, systempressure, and diffused optical density. As these conditions deviate fromOEC in response to microorganism growth steps must be taken to maintainOEC across these parameters. For example, if a Gen 7 cyanobacterialculture has tripled in density over a 48 hour period (observable throughincrease in DOD and Turbidity) and Ammonia has decreased to 0 ppm then adiluted addition of concentrated fertilizer should be added to returnAmmonia to OEC. Additionally, for example, if a Gen 7 culture has beenrun continuously for 3 months and in response to multiple feedings and abuildup of digestive waste pH has increased off OEC to 8.4 then adiluted addition of organic acid (i.e. citric acid) should be added toreturn pH to that culture's OEC for pH.

As exponential growth of photosynthetic microorganisms continues, DODwill continue to increase in proportion with culture density. Whenculture density has doubled three times from the point of its 10%inoculation it is at an ideal point to provide seed inoculation to otherreactors. At a 10% level of inoculation, the culture can be used to seed9 other reactors of comparable size with enough mass left over toself-inoculate its own restart. At a given point culture density willreach a level where the reactor primary fluid can no longer hold theculture in suspension. This point is identified by the increased levelsof accumulation/agglomeration in low flow points along the reactorbottom and sides in the presence of OEC. The point at which the systemtips toward this condition will be referred to as Peak Suspended Mass(PSM). When PSM is achieved the culture must be put through selectiveextraction, comprehensive extraction or used to seed other reactors ifgrowth rates are to be maintained. Time to PSM and culture density atPSM vary widely depending on strain and OEM.

D. Selective Extraction of Mature Cells

Prior to the point when PSM is achieved, one of two extractionconfigurations must be established: 1.) Gen 7 OutletValve=>Pump=>Filter=>Gen 7 Return; or 2.) Gen 7 OutletValve=>Filter=>Pump=>Gen 7 Return. The configuration is determined basedon the characteristics of the strain being filtered. The effectivenessof selective filtration is based on the size variance between mature andimmature cells in the given strain(s) and proper selection of the porediameter of the filtration medium. Filtration pore diameter should begreater than the diameter of the immature cells and less than thediameter of the mature cells by a preferred margin of >25%. Once one ofthe above configurations is set and PSM is achieved the following stepscan be taken: a.) Gen 7 outlet valve moved from CLOSED to OPEN; b.) oncepump is primed, pump moved from OFF to ON; c.) once filter bag is fulland air pressure has equalized across filter bag, filter cap can bemoved from CLOSED to OPEN; d.) optional: if the filter has an installedoutlet valve on its return line that valve should be moved from CLOSEDto OPEN before the pump and filter are moved to their engagedconfiguration. At no point should filter pressure exceed 15 psi, 10 psifor smaller diameter bags (<5 microns). Filtration should be monitoredclosely with new strains as filtration time varies widely with strain.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,and so forth used in the specification and claims are to be understoodas being modified in all instances by the term “about.” Accordingly,unless indicated to the contrary, the numerical parameters set forth inthe specification and attached claims are approximations that may varydepending upon the desired properties sought to be obtained by thepresent disclosure. At the very least, and not as an attempt to limitthe application of the doctrine of equivalents to the scope of theclaims, each numerical parameter should at least be construed in lightof the number of reported significant digits and by applying ordinaryrounding techniques.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the disclosure are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements.

The terms “a,” “an,” “the” and similar referents used in the context ofdescribing the disclosure (especially in the context of the followingclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.Recitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein isintended merely to better illuminate the disclosure and does not pose alimitation on the scope of the disclosure otherwise claimed. No languagein the specification should be construed as indicating any non-claimedelement essential to the practice of the disclosure.

Groupings of alternative elements or embodiments of the disclosuredisclosed herein are not to be construed as limitations. Each groupmember can be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. It isanticipated that one or more members of a group can be included in, ordeleted from, a group for reasons of convenience and/or patentability.When any such inclusion or deletion occurs, the specification is deemedto contain the group as modified thus fulfilling the written descriptionof all Markush groups used in the appended claims.

Certain embodiments of this disclosure are described herein, includingthe best mode known to the inventors for carrying out the disclosure. Ofcourse, variations on these described embodiments will become apparentto those of ordinary skill in the art upon reading the foregoingdescription. The inventor expects skilled artisans to employ suchvariations as appropriate, and the inventors intend for the disclosureto be practiced otherwise than specifically described herein.Accordingly, this disclosure includes all modifications and equivalentsof the subject matter recited in the claims appended hereto as permittedby applicable law. Moreover, any combination of the above-describedelements in all possible variations thereof is encompassed by thedisclosure unless otherwise indicated herein or otherwise clearlycontradicted by context.

Specific embodiments disclosed herein can be further limited in theclaims using, consisting of, or and consisting essentially, of language.When used in the claims, whether as filed or added per amendment, thetransition term “consisting of” excludes any element, step, oringredient not specified in the claims. The transition term “consistingessentially of” limits the scope of a claim to the specified materialsor steps and those that do not materially affect the basic and novelcharacteristic(s). Embodiments of the disclosure so claimed areinherently or expressly described and enabled herein.

It is to be understood that the embodiments of the disclosure disclosedherein are illustrative of the principles of the present disclosure.Other modifications that can be employed are within the scope of thedisclosure. Thus, by way of example, but not of limitation, alternativeconfigurations of the present disclosure can be utilized in accordancewith the teachings herein. Accordingly, the present disclosure is notlimited to that precisely as shown and described.

While the present disclosure has been described and illustrated hereinby references to various specific materials, procedures and examples, itis understood that the disclosure is not restricted to the particularcombinations of materials and procedures selected for that purpose.Numerous variations of such details can be implied as will beappreciated by those skilled in the art. It is intended that thespecification and examples be considered as exemplary, only, with thetrue scope and spirit of the disclosure being indicated by the followingclaims. All references, patents, and patent applications referred to inthis application are herein incorporated by reference in their entirety.

1. A photobioreactor for cultivation and/or propagation of a photosynthetic organism comprising: a.) a vessel having a wall defining an interior vessel volume; and b.) a lamp assembly positioned within the interior vessel volume, wherein the lamp assembly comprises: a plurality of circuit boards, each comprising at least three edges, arranged in a substantially spherical shape defining an interior lamp assembly volume, wherein the plurality of circuit boards comprise a first planar surface in contact with the interior lamp assembly volume and an opposing second planar surface comprising light emitting diodes (LEDs); and a barrier that surrounds the plurality of circuit boards forming the substantially spherical shape.
 2. The bioreactor of claim 1, wherein the vessel is cylindrical and comprises a cylindrical wall, an upper wall, and a lower wall each defining the interior tank volume.
 3. The bioreactor of claim 1, wherein the vessel is substantially spherical.
 4. The bioreactor of claim 1, wherein the vessel comprises a hole for a gas inlet.
 5. The bioreactor of claim 1, wherein the vessel comprises a hole for a gas outlet.
 6. The bioreactor of claim 1, wherein the vessel comprises a hole for wiring the light source.
 7. The bioreactor of claim 1, wherein the lamp assembly is positioned in the center of the vessel.
 8. The bioreactor of claim 1, wherein two or more lamp assemblies are positioned in the vessel.
 9. The bioreactor of claim 8, wherein the two or more lamp assemblies are positioned at different heights in the vessel.
 10. The bioreactor of claim 1, wherein three or more lamp assemblies are positioned in the vessel.
 11. The bioreactor of claim 10, wherein the three or more lamp assemblies are positioned at different heights in the vessel.
 12. The bioreactor of claim 11, wherein the three or more lamp assemblies are positioned in a helical arrangement in the vessel.
 13. A lamp assembly for use in cultivation and/or propagation of a photosynthetic organism, the lamp assembly comprising: a plurality of circuit boards, each comprising at least three edges, arranged in a substantially spherical shape defining an interior lamp assembly volume, wherein the plurality of circuit boards comprise a first planar surface in contact with the interior lamp assembly volume and an opposing second planar surface comprising light emitting diodes (LEDs); and a barrier that surrounds the plurality of circuit boards forming the substantially spherical shape.
 14. The light source of claim 13, wherein the substantially spherical shaped arrangement of the planar circuit boards has a side devoid of at least one circuit board to permit electrical connectivity.
 15. The light source of claim 13, wherein the circuit boards comprise two or more tabs around their perimeter that form one or more notches that permit the circuit boards to interlock.
 16. The light source of claim 13, wherein the circuit boards are pentagon shaped.
 17. The light source of claim 16, wherein eleven pentagons are joined to form a dodecahedron devoid of one side.
 18. The light source of claim 13, wherein the circuit boards are triangular shaped.
 19. The light source of claim 18, wherein twenty triangles are joined to form an icosahedron devoid of one side.
 20. The light source of claim 13, wherein the circuit boards comprise red, white, and blue LEDs.
 21. The light source of claim 13, wherein the red, white, and blue LEDs are positioned adjacent to an LED of opposing color.
 22. The light source of claim 13, wherein the barrier is plastic.
 23. The light source of claim 13, wherein the barrier is substantially spherical.
 24. The light source of claim 22, wherein the plastic permits transmission of light.
 25. The light source of claim 22, wherein the barrier has an open end to permit electrical connectivity.
 26. The light source of claim 22, wherein a void between the barrier and the circuit boards comprises a fluid for dispersal of heat.
 27. The light source of claim 26, wherein the fluid is mineral oil.
 28. A method of producing docosahexaenoic acid (DHA), the method comprising: a.) providing one or more photosynthetic organisms comprising enzymes for generating DHA; b.) adding the photosynthetic organisms to a vessel of a bioreactor comprising a liquid growth media; c.) contacting the one or more photosynthetic organisms with light emitted from a lamp assembly, wherein the lamp assembly comprises: a plurality of circuit boards, each comprising at least three edges, arranged in a substantially spherical shape defining an interior lamp assembly volume, wherein the plurality of circuit boards comprise a first planar surface in contact with the interior lamp assembly volume and an opposing second planar surface comprising light emitting diodes (LEDs); and a barrier that surrounds the plurality of circuit boards forming the substantially spherical shape; and d.). producing DHA from the one or more photosynthetic organisms.
 29. The method of claim 28, wherein the one or more photosynthetic organisms are algae.
 30. A method for storage of a light energy, the method comprising a.) providing one or more photosynthetic organisms comprising enzymes for generating one or more compounds from a light energy; b.) adding the one or more photosynthetic organisms to a tank of a bioreactor comprising a liquid growth media; c.) contacting the one or more photosynthetic organisms with light emitted from a lamp assembly, wherein the lamp assembly comprises: a plurality of circuit boards, each comprising at least three edges, arranged in a substantially spherical shape defining an interior lamp assembly volume, wherein the plurality of circuit boards comprise a first planar surface in contact with the interior lamp assembly volume and an opposing second planar surface comprising light emitting diodes (LEDs); and a barrier that surrounds the plurality of circuit boards forming the substantially spherical shape; and d.). producing one or more compounds from the light energy. 